The Effect And Mechanism Of Inhibiting Glutamine Metabolism In Triple Negative Breast Caner

Objectives:Metabolic reprogramming is an essential characteristic for malignant tumor.More and more evidences showed that the requirements for glutamine undergo changes to supply energy-generating and biosynthetic precursors for nucleotide biosynthesis,amino acid production,fatty acid synthesis and provide reducing equivalents to maintain redox status.Targeting glutamine metabolism present a potential approach of cancer therapy.CB-839 is an orally bioavailable inhibitor of the glutaminase 1 which is the key enzyme of glutaminolysis.Several recent studies showed synergetic lethality when tumor cells were treated with GLS1 inhibitor plus other drug.In this study,we investigated the correlation between subtypes of breast cancer,clinical prognosis and GLS1 expression.We studied the antitumor effect of GLS inhibitor in triple negative breast cancer and aimed at exploring the potential candidate for combination therapy.Methods:（1）This study analyzed the expression of GLS1 in breast cancer tissue samples by tissue microarray,immunohistochemistry and investigated the correlation between GLS1 and clinical characteristics.（2）Western Blot was performed to study the expression of GLS1 and MYC,and we utilized ChIP-sequence to disclose a novel role of MYC in regulating GLS transcription by directly binding on promotor regions of GLS.（3）Cell cultured under glutamine deprivation could reflect the dependence of glutaminolysis.（4）Cell viability assay was performed to investigate the proliferation inhibition of CB-839 on breast cancer cells.（5）Xenograft mice were treated with CB-839 and[U-¹³C]glutamine.Metabolite fractions were analyzed by LC-MS.（6）Cell viability assay and clonogenic assay were performed to evaluate the synergetic effect of CB-839 combined with carboplatin.（7）DNA fiber assay was performed to evaluate replication profiles.（8）Flow cytometry analysis were used to interrogated cell cycle arrest.（9）Xenograft models in immunodeficient animals were utilized to test the anti-tumor effect of single drug or CB-839 combined with carboplatin.And immunohistochemistry was performed to detect molecular signaling.Results:（1）We quantified the GLS expression in human breast samples,by tissue microarray,showed that high GLS expressed in triple negative breast cancer and positivity correlates with higher grade and poor patient survival.（2）The expression of GLS1 was positively associated with the expression of MYC.And MYC regulate GLS transcription by directly binding on promotor regions of GLS.（3）Triple negative breast cancer cell lines showed high expression of GLS and addiction to glutamine.GLS inhibitor CB-839impaired the proliferation of triple negative breast cancer cell lines.（4）CB-839 inhibited glutaminolysis and reductive carboxylation and the metabolites were decreased in triple negative breast cancer xenografts.（5）Synergistic effect of CB-839 and carboplatin was observed in GLS1 high expression cell lines.（6）Combination of CB-839 and carboplatin inhibited tumor proliferation by reducing the level of reduced glutathione,inducing ROS level,DNA damage and cell cycle arrest.（7）CB-839 combined with carboplatin resulted in prolonged disease remission and reduced tumor growth in vivo.Synergistic induction of anti-proliferation and DNA damage by combination treatment was evident in xenograft tumor.Conclusion:High level of GLS1 indicates a poor prognosis.Triple negative breast cancer have upregulated glutaminase compared with other subtypes of breast cancer and higher sensitivity towards glutaminase inhibitor CB-839.CB-839 inhibited glutaminolysis and reductive carboxylation,which resulted in the reduction of metabolites.Combination of GLS inhibitor and carboplatin caused further reduction of reduced glutathione and accumulation of ROS,which resulted in synergistic antitumor effect.Combination treatment significantly induced DNA damage,cell cycle arrest and impaired DNA replication.Together,our finding suggests that a combined approach of using GLS inhibitor and administering common chemotherapeutic agent carboplatin is promising for triple negative breast cancer treatment.